RESUMO
Specific information about how telomerase acts in vivo is necessary for understanding telomere dynamics in human tumor cells. Our results imply that, under homeostatic telomere length-maintenance conditions, only one molecule of telomerase acts at each telomere during every cell division and processively adds â¼60 nt to each end. In contrast, multiple molecules of telomerase act at each telomere when telomeres are elongating (nonequilibrium conditions). Telomerase extension is less processive during the first few weeks following the reversal of long-term treatment with the telomerase inhibitor Imetelstat (GRN163L), a time when Cajal bodies fail to deliver telomerase RNA to telomeres. This result implies that processing of telomerase by Cajal bodies may affect its processivity. Overexpressed telomerase is also less processive than the endogenously expressed telomerase. These findings reveal two major distinct extension modes adopted by telomerase in vivo.
Assuntos
Homeostase , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Western Blotting , Linhagem Celular Tumoral , Corpos Enovelados/metabolismo , Fase G1 , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Modelos Genéticos , Oligonucleotídeos/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase S , Telomerase/antagonistas & inibidores , Telomerase/genéticaRESUMO
Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for antioxidant and anti-inflammation enzymes that binds to its endogenous inhibitor protein, Kelch-like ECH (erythroid cell-derived protein with CNC homology)-associated protein 1, in the cytoplasm under normal conditions. Various endogenous or environmental oxidative stresses, such as ionizing radiation (IR), can disrupt the Nrf2-Kelch-like ECH-associated protein 1 complex. This allows Nrf2 to translocate from the cytoplasm into the nucleus to induce transcription of heme oxygenase-1 and other cytoprotective enzymes through binding to antioxidant responsive elements. However, how Nrf2 protects cells from IR-induced damage remains unclear. Here, we report that Nrf2 activation by the synthetic triterpenoids, bardoxolone methyl (BARD) and 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide, protects colonic epithelial cells against IR-induced damage, in part, by enhancing signaling of the DNA damage response. Pretreatment with BARD reduced the frequency of both G1 and S/G2 chromosome aberrations and enhanced the disappearance of repairosomes (C-terminal binding protein interacting protein, Rad51, and p53 binding protein-1 foci) after IR. BARD protected cells from IR toxicity in a Nrf2-dependent manner. The p53 binding protein-1 promoter contains three antioxidant responsive elements in which Nrf2 directly binds following BARD treatment. In addition, 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide provided before exposure to a lethal dose of whole-body irradiation protected WT mice from DNA damage and acute gastrointestinal toxicity, which resulted in improved overall survival. These results demonstrate that Nrf2 activation by synthetic triterpenoids is a promising candidate target to protect the gastrointestinal tract against acute IR in vitro and in vivo.
Assuntos
Colo/efeitos da radiação , Dano ao DNA , Mucosa Intestinal/efeitos da radiação , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Transformada , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Radiação IonizanteRESUMO
INTRODUCTION: The clinical benefit of the anti-CTLA-4 monoclonal antibody (mAb) ipilimumab has been well established but limited by immune-related adverse events, especially when ipilimumab is used in combination with anti-PD-(L)1 mAb therapy. To overcome these limitations, we have developed XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 mAb. METHODS: XTX101 consists of an anti-human CTLA-4 mAb covalently linked to masking peptides that block the complementarity-determining regions, thereby minimizing the mAb binding to CTLA-4. The masking peptides are designed to be released by proteases that are typically dysregulated within the tumor microenvironment (TME), resulting in activation of XTX101 intratumorally. Mutations within the Fc region of XTX101 were included to enhance affinity for FcγRIII, which is expected to enhance potency through antibody-dependent cellular cytotoxicity. RESULTS: Biophysical, biochemical, and cell-based assays demonstrate that the function of XTX101 depends on proteolytic activation. In human CTLA-4 transgenic mice, XTX101 monotherapy demonstrated significant tumor growth inhibition (TGI) including complete responses, increased intratumoral CD8+T cells, and regulatory T cell depletion within the TME while maintaining minimal pharmacodynamic effects in the periphery. XTX101 in combination with anti-PD-1 mAb treatment resulted in significant TGI and was well tolerated in mice. XTX101 was activated in primary human tumors across a range of tumor types including melanoma, renal cell carcinoma, colon cancer and lung cancer in an ex vivo assay system. CONCLUSIONS: These data demonstrate that XTX101 retains the full potency of an Fc-enhanced CTLA-4 antagonist within the TME while minimizing the activity in non-tumor tissue, supporting the further evaluation of XTX101 in clinical studies.
Assuntos
Antineoplásicos , Melanoma , Humanos , Camundongos , Animais , Antígeno CTLA-4 , Ipilimumab/uso terapêutico , Antineoplásicos/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Melanoma/tratamento farmacológico , Modelos Animais de Doenças , Camundongos Transgênicos , Peptídeos/uso terapêutico , Microambiente TumoralRESUMO
Normal human colonic epithelial cells (HCECs) are not immortalized by telomerase alone but also require CDK4. Some human cell types growth-arrest due to stress- or aberrant signaling-induced senescence (stasis). Stasis represents the consequences of growth conditions culture that are inadequate to maintain long-term proliferation. Overexpressed CDK4 titers out p16 and allows cells to ignore the growth arrest signals produced by stasis. To identify factors contributing to the inadequate culture environment, we used a 62,000-member shRNA library to knock down factors cooperating with human telomerase reverse transcriptase (hTERT) in the immortalization of HCECs. Knockdown of Klotho gamma (KLG; also known as KLPH and LCTL) allowed hTERT to immortalize HCECs. KLG is one isoform of the Klotho family of factors that coordinate interaction between different FGF ligands and the FGF receptor. We also found that knockdown of KLG induced another member of the Klotho family, Klotho beta (KLB). Induction of KLB was maintained and could activate ERK1/2 in immortalized cells. Supplementation of the culture medium with the KLB ligand FGF19 had a similar effect on hTERT-expressing HCECs as knockdown of KLG regarding both immortalization and down-regulation of the tumor suppressor Klotho alpha. Together, these data suggest that KLB is an important regulator in the immortalization of HCECs by facilitating FGF19 growth factor signaling.
Assuntos
Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Colo/citologia , Células Epiteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/metabolismo , RNA Interferente Pequeno/fisiologia , Western Blotting , Linhagem Celular , Fatores de Crescimento de Fibroblastos/farmacologia , Glucuronidase/genética , Humanos , Proteínas Klotho , RNA Interferente Pequeno/genética , Telomerase/genética , Telomerase/metabolismoRESUMO
BACKGROUND & AIMS: Long-term propagation of human colonic epithelial cells (HCEC) of adult origin has been a challenge; currently used HCEC lines are of malignant origin and/or contain multiple cytogenetic changes. We sought to immortalize human colon biopsy-derived cells expressing stem cell markers and retaining multilineage epithelial differentiation capability. METHODS: We isolated and cultured cells from biopsy samples of 2 patients undergoing routine screening colonoscopy. Cells were immortalized by expression of the nononcogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of human telomerase (hTERT) and maintained for more than 1 year in culture. RESULTS: The actively proliferating HCECs expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. Upon growth arrest, cells assumed a cuboidal shape, decreased their mesenchymal features, and expressed markers of colonic epithelial cells such as cytokeratin 18, zonula occludens-1, mucins-1 and -2, antigen A33, and dipeptidyl peptidase 4. Immortalized cells expressed stem cell markers that included LGR5, BMI1, CD29, and CD44. When placed in Matrigel in the absence of a mesenchymal feeder layer, individual cells divided and formed self-organizing, cyst-like structures; a subset of cells exhibited mucin-2 or polarized villin staining. CONCLUSIONS: We established immortalized HCECs that are capable of self-renewal and multilineage differentiation. These cells should serve as valuable reagents for studying colon stem cell biology, differentiation, and pathogenesis.
Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Colo/metabolismo , Células Epiteliais/metabolismo , Células-Tronco/metabolismo , Biópsia , Linhagem Celular , Proliferação de Células , Forma Celular , Colágeno/metabolismo , Colo/ultraestrutura , Colonoscopia , Quinase 4 Dependente de Ciclina/genética , Combinação de Medicamentos , Células Epiteliais/ultraestrutura , Humanos , Cariotipagem , Laminina/metabolismo , Proteoglicanas/metabolismo , Células-Tronco/ultraestrutura , Telomerase/genética , Fatores de Tempo , TransfecçãoRESUMO
Deep learning has emerged as the technique of choice for identifying hidden patterns in cell imaging data but is often criticized as "black box." Here, we employ a generative neural network in combination with supervised machine learning to classify patient-derived melanoma xenografts as "efficient" or "inefficient" metastatic, validate predictions regarding melanoma cell lines with unknown metastatic efficiency in mouse xenografts, and use the network to generate in silico cell images that amplify the critical predictive cell properties. These exaggerated images unveiled pseudopodial extensions and increased light scattering as hallmark properties of metastatic cells. We validated this interpretation using live cells spontaneously transitioning between states indicative of low and high metastatic efficiency. This study illustrates how the application of artificial intelligence can support the identification of cellular properties that are predictive of complex phenotypes and integrated cell functions but are too subtle to be identified in the raw imagery by a human expert. A record of this paper's transparent peer review process is included in the supplemental information. VIDEO ABSTRACT.
Assuntos
Aprendizado Profundo , Melanoma , Animais , Inteligência Artificial , Humanos , Camundongos , Redes Neurais de ComputaçãoRESUMO
CD137 (4-1BB) is a member of the TNFR superfamily that represents a promising target for cancer immunotherapy. Recent insights into the function of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as critical features, and these observations help explain the limited activity and toxicity seen with clinically tested CD137 agonists. Here, we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 that engages a unique epitope that is shared by human, cynomolgus monkey, and mouse and is associated with a differentiated pharmacology and toxicology profile. In vitro, CTX-471 increased IFN-γ production by human T cells in an Fcγ receptor-dependent (FcγR-dependent) manner, displaying an intermediate level of activity between 2 clinical-stage anti-CD137 antibodies. In mice, CTX-471 exhibited curative monotherapy activity in various syngeneic tumor models and showed a unique ability to cure mice of very large (~500 mm3) tumors compared with validated antibodies against checkpoints and TNFR superfamily members. Extremely high doses of CTX-471 were well tolerated, with no signs of hepatic toxicity. Collectively, these data demonstrate that CTX-471 is a unique CD137 agonist that displays an excellent safety profile and an unprecedented level of monotherapy efficacy against very large tumors.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Mapeamento de Epitopos , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Imunoterapia/efeitos adversos , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Macaca fascicularis , Camundongos , Camundongos Nus , Neoplasias/imunologia , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Emerging evidence suggests that crosstalk between glioma cells and the brain microenvironment may influence brain tumor growth. To date, known reciprocal interactions among these cells have been limited to the release of paracrine factors. Combining a genetic strategy with longitudinal live imaging, we find that individual gliomas communicate with distinct sets of non-glioma cells, including glial cells, neurons, and vascular cells. Transfer of genetic material is achieved mainly through extracellular vesicles (EVs), although cell fusion also plays a minor role. We further demonstrate that EV-mediated communication leads to the increase of synaptic activity in neurons. Blocking EV release causes a reduction of glioma growth in vivo. Our findings indicate that EV-mediated interaction between glioma cells and non-glioma brain cells alters the tumor microenvironment and contributes to glioma development.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Comunicação Celular , Vesículas Extracelulares/metabolismo , Glioma/patologia , Animais , Astrócitos/patologia , Encéfalo/fisiopatologia , Neoplasias Encefálicas/fisiopatologia , Fusão Celular , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Fenômenos Eletrofisiológicos , Vesículas Extracelulares/ultraestrutura , Glioma/fisiopatologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neurônios/patologia , Imagem com Lapso de TempoRESUMO
Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αß and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αß T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αß T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αß and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.
Assuntos
Antígenos CD/imunologia , Butirofilinas/antagonistas & inibidores , Butirofilinas/imunologia , Linfócitos Intraepiteliais/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/genética , Butirofilinas/genética , Feminino , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Soft agar anchorage-independent growth assays have been commonly used as an indicator of cellular transformation in cell culture. Protocols listed here are optimized to allow for all steps, including plasmid purification, virus production, transduction, and soft agar colony formation, to be performed in 96-well plates. These modifications decrease hands-on time, increase fidelity of the assay, and make it possible to screen 500-1000 short-hairpin RNAs (shRNA) in "one-shRNA-one-well" format in parallel. These protocols can also be used to conduct functional cDNA or CRISPR screens for modulators of anchorage-independent growth.
Assuntos
Adesão Celular , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Inativação Gênica , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/genética , Ágar/química , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
We screen ion channels and transporters throughout the genome to identify those required by human melanoma cells but not by normal human melanocytes. We discover that Mucolipin-1 (MCOLN1), which encodes the lysosomal cation channel TRPML1, is preferentially required for the survival and proliferation of melanoma cells. Loss of MCOLN1/TRPML1 function impairs the growth of patient-derived melanomas in culture and in xenografts but does not affect the growth of human melanocytes. TRPML1 expression and macropinocytosis are elevated in melanoma cells relative to melanocytes. TRPML1 is required in melanoma cells to negatively regulate MAPK pathway and mTORC1 signaling. TRPML1-deficient melanoma cells exhibit decreased survival, proliferation, tumor growth, and macropinocytosis, as well as serine depletion and proteotoxic stress. All of these phenotypes are partially or completely rescued by mTORC1 inhibition. Melanoma cells thus increase TRPML1 expression relative to melanocytes to attenuate MAPK and mTORC1 signaling, to sustain macropinocytosis, and to avoid proteotoxic stress.
Assuntos
Sistema de Sinalização das MAP Quinases , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Melanoma/metabolismo , Proteostase , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Masculino , Melanoma/genética , Melanoma/patologia , Camundongos , Fenótipo , Pinocitose , Canais de Potencial de Receptor Transitório/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Dose- and time-dependent effects of doxorubicin on telomerase activity (TA) and expression levels of hTERT, Bcl-2, Bcl-x(L) and Bax were investigated in doxorubicin-resistant and -sensitive MCF-7 cells. METHODS: Doxorubicin-resistant MCF-7/R was developed from sensitive MCF-7 breast carcinoma cell line and acquired resistance was demonstrated by XTT and mRNA analysis of MDR1 and MRP1 genes. Expression levels were determined by RT-PCR. Newly developed rapid and simple TRAP-silver staining assay was used to assess TA levels. RESULTS: Doxorubicin-selected MCF-7 cells were 107-fold resistant to the drug and overexpress MDR1 and MRP1 genes. 72 h doxorubicin incubation caused a decrease in TA in parallel with a small decrease in hTERT level in both sensitive and resistant cells. Bcl-2 expression level decreased upon doxorubicin application in sensitive cells. However, the Bcl-x(L)level increased in sensitive cells after 72 h of doxorubicin incubation. CONCLUSION: This report demonstrates the inhibitory effects of doxorubicin on TA in both resistant and sensitive MCF-7 cells possibly through modulation of the apoptotic pathway genes.
Assuntos
Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sensibilidade e EspecificidadeRESUMO
ETS transcription factors are commonly deregulated in cancer by chromosomal translocation, overexpression or post-translational modification to induce gene expression programs essential in tumorigenicity. Targeted destruction of these proteins may have therapeutic impact. Here we report that Ets-1 destruction is regulated by the deubiquitinating enzyme, Usp9x, and has major impact on the tumorigenic program of metastatic melanoma. Ets-1 deubiquitination blocks its proteasomal destruction and enhances tumorigenicity, which could be reversed by Usp9x knockdown or inhibition. Usp9x and Ets-1 levels are coincidently elevated in melanoma with highest levels detected in metastatic tumours versus normal skin or benign skin lesions. Notably, Ets-1 is induced by BRAF or MEK kinase inhibition, resulting in increased NRAS expression, which could be blocked by inactivation of Usp9x and therapeutic combination of Usp9x and MEK inhibitor fully suppressed melanoma growth. Thus, Usp9x modulates the Ets-1/NRAS regulatory network and may have biologic and therapeutic implications.
Assuntos
Carcinogênese/patologia , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Melanoma/tratamento farmacológico , Proteínas de Membrana/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
This is the first prospective study of a combination therapy involving a cardenolide and a MEK inhibitor for metastatic melanoma. Whereas BRAF mutant melanomas can exhibit profound responses to treatment with BRAF and MEK inhibitors, there are fewer options for BRAF wild-type melanomas. In preclinical studies, we discovered that cardenolides synergize with MEK inhibitor to promote the regression of patient-derived xenografts irrespective of BRAF mutation status. We therefore conducted a phase 1B study of digoxin 0.25 mg and trametinib 2 mg given orally once daily in 20 patients with advanced, refractory, BRAF wild-type melanomas. The most common adverse events were rash, diarrhea, nausea, and fatigue. The response rate was 4/20 or 20% with response durations of 2, 4, 6, and 8 months. The disease control rate (including partial responses and stable disease) was 13/20 or 65% of patients, including 5/6 or 83% of patients with NRAS mutant melanomas and 8/14 or 57% of NRAS wild-type melanomas. Patients with stable disease had disease control for 2, 2, 2, 4, 5, 6, 7, 10, and 10 months. Xenografts from four patients recapitulated the treatment responses observed in patients. Based on these pilot results, an expansion arm of digoxin plus MEK inhibitor is warranted for NRAS mutant metastatic melanoma patients who are refractory or intolerant of immunotherapy. KEY POINTS: Digoxin plus trametinib is well tolerated and achieves a high rate of disease control in BRAF wild-type metastatic melanoma patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Digoxina/administração & dosagem , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Melanoma/patologia , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Retratamento , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genomic diversity among melanoma tumors limits durable control with conventional and targeted therapies. Nevertheless, pathologic activation of the ERK1/2 pathway is a linchpin tumorigenic mechanism associated with the majority of primary and recurrent disease. Therefore, we sought to identify therapeutic targets that are selectively required for tumorigenicity in the presence of pathologic ERK1/2 signaling. By integration of multigenome chemical and genetic screens, recurrent architectural variants in melanoma tumor genomes, and patient outcome data, we identified two mechanistic subtypes of BRAFV600 melanoma that inform new cancer cell biology and offer new therapeutic opportunities. Subtype membership defines sensitivity to clinical MEK inhibitors versus TBK1/IKBKε inhibitors. Importantly, subtype membership can be predicted using a robust quantitative five-feature genetic biomarker. This biomarker, and the mechanistic relationships linked to it, can identify a cohort of best responders to clinical MEK inhibitors and identify a cohort of TBK1/IKBKε inhibitor-sensitive disease among nonresponders to current targeted therapy.Significance: This study identified two mechanistic subtypes of melanoma: (1) the best responders to clinical BRAF/MEK inhibitors (25%) and (2) nonresponders due to primary resistance mechanisms (9.9%). We identified robust biomarkers that can detect these subtypes in patient samples and predict clinical outcome. TBK1/IKBKε inhibitors were selectively toxic to drug-resistant melanoma. Cancer Discov; 7(8); 832-51. ©2017 AACR.See related commentary by Jenkins and Barbie, p. 799This article is highlighted in the In This Issue feature, p. 783.
Assuntos
Biomarcadores Tumorais/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Carcinogênese/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/classificação , Melanoma/patologia , Camundongos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
New therapies are required for melanoma. Here, we report that multiple cardiac glycosides, including digitoxin and digoxin, are significantly more toxic to human melanoma cells than normal human cells. This reflects on-target inhibition of the ATP1A1 Na(+)/K(+) pump, which is highly expressed by melanoma. MEK inhibitor and/or BRAF inhibitor additively or synergistically combined with digitoxin to induce cell death, inhibiting growth of patient-derived melanomas in NSG mice and synergistically extending survival. MEK inhibitor and digitoxin do not induce cell death in human melanocytes or haematopoietic cells in NSG mice. In melanoma, MEK inhibitor reduces ERK phosphorylation, while digitoxin disrupts ion gradients, altering plasma membrane and mitochondrial membrane potentials. MEK inhibitor and digitoxin together cause intracellular acidification, mitochondrial calcium dysregulation and ATP depletion in melanoma cells but not in normal cells. The disruption of ion homoeostasis in cancer cells can thus synergize with targeted agents to promote tumour regression in vivo.
Assuntos
Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Digitoxina/farmacologia , Digitoxina/uso terapêutico , Sinergismo Farmacológico , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , MAP Quinase Quinase 1/metabolismo , Masculino , Melanócitos , Melanoma/mortalidade , Melanoma/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Fosforilação , Cultura Primária de Células , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Pele/patologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mutations in the adenomatous polyposis coli (APC) gene are common in colorectal cancer (CRC), and more than 90% of those mutations generate stable truncated gene products. We describe a chemical screen using normal human colonic epithelial cells (HCECs) and a series of oncogenically progressed HCECs containing a truncated APC protein. With this screen, we identified a small molecule, TASIN-1 (truncated APC selective inhibitor-1), that specifically kills cells with APC truncations but spares normal and cancer cells with wild-type APC. TASIN-1 exerts its cytotoxic effects through inhibition of cholesterol biosynthesis. In vivo administration of TASIN-1 inhibits tumor growth of CRC cells with truncated APC but not APC wild-type CRC cells in xenograft models and in a genetically engineered CRC mouse model with minimal toxicity. TASIN-1 represents a potential therapeutic strategy for prevention and intervention in CRC with mutant APC.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Terapia de Alvo Molecular , Piperidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Proliferação de Células , Colesterol/química , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Feminino , Genes Supressores de Tumor , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Transgenes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The RASSF1A gene is one of the most frequently inactivated genes in over 30 different types of cancers (H. Donninger, M. D. Vos, and G. J. Clark, J. Cell Sci. 120:3163-3172, 2007, http://dx.doi.org/10.1242/jcs.010389). Despite the prevalence of RASSF1A silencing in human cancer, the mechanism by which RASSF1A functions as a tumor suppressor is not well understood. Characterization of the consequences of RASSF1A loss on epithelial cell proliferation revealed that RASSF1A expression suppresses both microRNA 21 (miR-21) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. The mechanism of the former is through restraint of SCF(ßTrCP)-dependent destruction of the repressor element 1 silencing transcription factor (REST) tumor suppressor and consequent inhibition of miR-21 promoter activation. The mechanism of the latter is through physical sequestration of MST2, which results in accumulation of inactivating S259 phosphorylation of RAF1. Whether or not inactivation of these RASSF1A regulatory relationships can unleash enhanced proliferative capacity is dependent upon the coupling of SCF(ßTrCP) and miR-21 to suppression of SKP2 protein translation and stability. Airway epithelial cultures retain this coupling and therefore respond to RASSF1A inactivation by p27-dependent cell cycle arrest. In contrast, colonic crypt-derived epithelial cells have uncoupled SCF(ßTrCP) from SKP2 and respond to RASSF1A inactivation by enhanced proliferation rates. These observations help account for context-specific molecular etiology of oncogenic transformation and suggest intervention strategies for recently developed SKP2 inhibitors.
Assuntos
Ciclo Celular/genética , Genes Supressores de Tumor , Oncogenes , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Masculino , MicroRNAs/metabolismo , Dados de Sequência Molecular , Proteínas Repressoras/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismoRESUMO
Studies of human cancer metastasis have been limited by a lack of experimental assays in which cancer cells from patients metastasize in vivo in a way that correlates with clinical outcome. This makes it impossible to study intrinsic differences in the metastatic properties of cancers from different patients. We recently developed an assay in which human melanomas readily engraft in nonobese diabetic/severe combined immunodeficient interleukin-2 receptor-γ chain null (NSG) mice. We show that melanomas from 25 patients exhibited reproducible differences in the rate of spontaneous metastasis after transplantation into NSG mice and that these differences correlated with clinical outcome in the patients. Stage IIIB/C melanomas that formed distant metastases within 22 months in patients also formed tumors that metastasized widely in NSG mice, whereas stage IIIB/C melanomas that did not form distant metastases within 22 to 50 months in patients metastasized more slowly in NSG mice. These differences in the efficiency of metastasis correlated with the presence of circulating melanoma cells in the blood of NSG mice, suggesting that the rate of entry into the blood is one factor that limits the rate of metastasis. The study of NSG mice can therefore yield information about the metastasis of human melanomas in vivo, in this case revealing intrinsic differences among stage III melanomas in their ability to circulate/survive in the blood and to metastasize.
Assuntos
Subunidade gama Comum de Receptores de Interleucina/deficiência , Melanoma/patologia , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Progressão da Doença , Humanos , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Medições Luminescentes , Camundongos , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Resultado do TratamentoRESUMO
Landmark cancer genome resequencing efforts are leading to the identification of mutated genes in many types of cancer. The extreme diversity of mutations being detected presents significant challenges to subdivide causal from coincidental mutations to elucidate how disrupted regulatory networks drive cancer processes. Given that a common early perturbation in solid tumor initiation is bypass of matrix-dependent proliferation restraints, we sought to functionally interrogate colorectal cancer candidate genes (CAN-genes) to identify driver tumor suppressors. We have employed an isogenic human colonic epithelial cell (HCEC) model to identify suppressors of anchorage-independent growth by conducting a soft agar-based short hairpin RNA (shRNA) screen within the cohort of CAN-genes. Remarkably, depletion of 65 of the 151 CAN-genes tested collaborated with ectopic expression of K-RAS(V12) and/or TP53 knockdown to promote anchorage-independent proliferation of HCECs. In contrast, only 5 of 362 random shRNAs (1.4%) enhanced soft agar growth. We have identified additional members of an extensive gene network specifying matrix-dependent proliferation, by constructing an interaction map of these confirmed progression suppressors with approximately 700 mutated genes that were excluded from CAN-genes, and experimentally verifying soft agar growth enhancement in response to depletion of a subset of these genes. Collectively, this study revealed a profound diversity of nodes within a fundamental tumor suppressor network that are susceptible to perturbation leading to enhanced cell-autonomous anchorage-independent proliferative fitness. Tumor suppressor network fragility as a paradigm within this and other regulatory systems perturbed in cancer could, in large part, account for the heterogeneity of somatic mutations detected in tumors.